1. Field of the Invention
This invention relates generally to the transfer of molecules into a plant, and specifically to a method of introducing molecules such as polypeptides or polynucleotides into an intact plant or plant tissue using electroporation.
2. Description of Related Art
The ability to alter the genetic composition of a living cell by transformation is one of the core technologies in biotechnology. By manipulating the genetic repertoire of a cell, one can produce large amounts of a desirable protein product. One of the primary limitations to the alteration of cells has been the technology to carry out the transformation.
Genetically engineered plants are useful for the production of vaccines against human diseases, ranging from tooth decay to life-threatening infections such as bacterial diarrhea, cholera, and AIDS. It may even be possible to produce vaccines in plants which are then eaten as part of the normal diet. Such vaccines might be cheaper than those now available, because plants are easier to grow in large quantities than are the cultured animal cells which are currently being used to make most vaccines.
Most commonly, DNA transfer into plant cells has been accomplished by preparation of protoplasts which are subsequently treated with a DNA-containing solution which is taken up by the protoplast. As plant regeneration from protoplasts has generally been limited to a relatively small number of genotypes for various species, it has been difficult to develop a generally effective protoplast based procedure. Therefore, other approaches have been recently explored.
Typically, means for delivery of DNA into living cells include cellular uptake of DNA precipitates, microinjection of DNA into a single cell, electrofusion, insertion of DNA into cells by micro-projectiles coated with DNA, and cellular uptake of DNA from the surrounding solution following exposure of the cell to a strong electric pulse (i.e., electroporation).
Micro-injection of DNA into single cells is inefficient and tedious, and only a limited number of cells can be treated at one time. Electro-fusion is a means by which exogenous genetic material is introduced into a host plant (U.S. Pat. No. 4,832,814). The insertion of genetic material is accomplished by either permeabilizing the cell membrane to allow entry of genetic material or fusing the host cell with a cell containing the genetic material of interest. Electro-fusion has many limitations and does not work for all plant cells (see U.S. Pat. No. 4,822,470).
Insertion of DNA into cells using DNA-coated micro-projectiles (U.S. Pat. No. 4,945,050) has also been utilized for genetically modifying plants. The exposure to gas and debris from an explosive event arises from the need to use an explosion to achieve the high degree of acceleration required to give the micro-projectiles the requisite kinetic energy to pierce the cell. These conditions impose severe limitations to applying the projectile method to cells in an intact plant or organism. The high velocity ballistic penetration by small particles with the nucleic acid either within the matrix of small beads or particles, or on the surface (Klein, et al., Nature, 327:70, 1987). Although, typically only a single introduction of a new nucleic acid segment is required, this method particularly provides for multiple introductions. However, this technology requires that plants go through a tissue culture stage, and again the conditions for regeneration of whole plants from plant cells are not without problems.
Cellular uptake of DNA following exposure of the cell to an electric pulse is accomplished often in surroundings which are not too far from physiological, and often for an amount of time on the order of milliseconds or less. The target cells are typically required to be placed in a cavity (e.g., a cuvette). Most electro-transformation is carried out on cells in suspension after dissociating the tissue to single cells or small aggregates of cells before treating.
Direct transfer of genes into cells or intact plant leaves or tissues by electroporation, has required the addition of chemicals such as spermine, spermidine or lipophilic molecules such as Lipofectin.RTM. (see for example, U.S. Pat. No. 5,286,634). Most recently, Chowrira, et al. (Molecular Biotechnology, 3:17, 1995) showed studies which demonstrated that leguminous plants could be transformed by electroporation of DNA into intact nodal meristems in planta, resulting in transient expression and stable integration of transgenes, when DNA is first mixed with lipofectin reagent. The study showed that the amount of lipofectin added to the plasmid DNA was critical for the introduction of DNA to the plant cells.
Transient expression of gus gene (.beta.-glucuronidase) and anthocyanin was obtained in immature embryos by electroporation of DNA in a spermidine-containing electroporation solution (Songstad, et al., Plant Cell Tissue and Organ Culture, 33:195, 1993). NPTII (neomycin phosphotransferase II) gene expression was also greatly enhanced by transformation of DNA in buffer containing spermidine, which delays lysis of protoplasts and inhibits the activity of nucleases (Dekeyser, et al., The Plant Cell, 2:591, 1990). In contrast, transformation in the absence of spermidine resulted in extremely low if any gene expression. Spermine and lipofectin containing buffers were also utilized for transferring DNA via electroporation to intact cowpea seed-derived embryos (Akella and Lurquin, Plant Cell Reports, 12:110, 1993).
Monocot cells were transformed in the absence of cell wall degrading enzymes by electroporation of either single cell suspensions or cell aggregates, both from prior dissociation of plant tissue (Gobel, et al., WO93/21335). Therefore, in vitro cell culture was required following treatment.
There remains a need for a method of introducing molecules into plants which avoids the problems associated with regeneration from protoplasts and which allows many cells to be transformed at one time, in the absence of chemicals.